Published: October 29th, 2013
In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.
Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.
The purification of recombinant proteins is crucial to address fundamental questions in biochemistry. Conventional ways of protein purification like ion exchange chromatography and size exclusion chromatography rely on physical properties of the target protein such as its isoelectric point and charge or size, respectively. The latter protein characteristics are shared by a variety of proteins, which increases considerably the chance of contaminating proteins in conventional protein purification strategies. This problem may be circumvented with the use of multiple purification columns, which is time consuming. At the same time, the latter chromatography methods demand ....
1. Production of Recombinant Baculovirus
Baculoviruses are generated using the Bac-to-Bac Baculovirus Expression System of Invitrogen mainly in accordance with the manufacturer's protocol with only slight modifications:
In order to illustrate the efficiency of the GST-His purification protocol, we purified Rec14, a S. pombe protein of 32.9 kDa. The Rec14 cDNA was cloned in our modified pFastBac1 vector allowing the additions of GST- and His-tags at the N- and C- termini, respectively (Figure 1A). Recombinant baculovirus were then prepared and used to infect SF9 infected cells for protein expression. The soluble cell lysates were incubated with GST beads and bound-proteins were eluted by cleaving the GST with Pr.......
The GST-His purification protocol presented here is suitable for the purification of a broad range of sizes of recombinant proteins: We successfully purified LiRAD51 (41kDa), piBRCA2 (120kDa), PALB2 (130kDa), and an unstable high molecular weight protein of Leishmania infantum: LiBRCA2 (125kDa)6,9. Moreover, the proteins purified with this protocol were biochemically active6. The success of this method solely depends on the expression, solubility and stability of th.......
We thank Anne-Marie Dion-Cote for discussions leading to the development of the method. J.K. and R.B. are FQNRT doctoral scholars, M.-M.G. is a Vanier CIHR scholar, and J.-Y.M. is a FRSQ senior researcher. This work was supported by funds from the Natural Sciences and Engineering Research Council to J-Y. M.....
|Name of the reagent
|E.Coli DH10Bac (competent)
|Bac-to-Bac Baculovirus Expression System
|Maxi Prep Kit
|Solution I and II
|Ultra Pure Bluo-Gal
|Sf9 infected cells
|Grace's infected medium supplemented
|Fetal Bovine Serum characterized
|Glutathione Sepharose resin
|Protease inhibitor cocktail
|Production in house
|Dounce homogenizer (tight)
|Sonicator (Fisher dismembrator)
|Dialysis bag (50 mm)
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